This invention relates to an inducible or regulatable retroviral vector for the controlled delivery of a gene product and in particular to a tetracycline regulated retroviral vector for the controlled delivery of a gene product.
The introduction of heterologous genes into cultured mammalian cells or tissues is fundamental for understanding the biochemistry, genetics and function(s) of genes and gene products. Much of the understanding of biological processes has been determined by transfection, electroporation, or viral delivery of genes into cultured cells or tissues. More recently, gene therapy approaches have emphasized the need for gene delivery vectors that can efficiently introduce, and control the expression of heterologous genes in mammalian cells.
An important feature of any gene delivery system is the ability to regulate the expression of the delivered gene. This is important in situations where the translation product of the delivered gene is being examined for its functional role in cell biology and in therapeutic situations where the gene product is toxic or must be maintained at appropriate levels. Ideally, regulation of expression of the delivered gene should be in an ON or OFF manner. Where when turned ON, gene expression should be induced and when turned OFF gene expression should be silenced.
The tetracycline-controlled transactivator responsive promoter (Tet system) is a prokaryotic inducible promoter system which has been adapted for use in mammalian cells (Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci USA, 89, 5547-5551; Gossen, M., Bonin, A. L. & Bujard, H. (1993) TIBS 18, 471-475). In the previously developed Tet system, there are two components, each of which is carried on a separate plasmid. One component, the "response unit", is composed of the E. coli-derived tetracycline resistance operon regulatory elements (tetO) embedded within a minimal CMV promoter. The second component, the "regulator unit", encodes a transactivator hybrid protein (tTA) composed of the tetracycline repressor (tetR) fused to the herpes simplex virus (HSV) transactivator protein, VP16. Expression of a gene inserted downstream of the tetO/minimal CMV promoter in the response unit is highly dependent on tTA which binds tetO sequences through its tetR domain and recruits positively acting cellular transcription factors through its VP16 domain.
Gene expression is inhibited by the addition of tetracycline, which binds the transactivator protein, causing it to dissociate from the tetO/minimal CMV promoter and leading to cessation of gene transcription.
Although the Tet system has proven to be an important tool for examining the effects of genes on cells, it is limited in its applications for the following reasons:
1) For the Tet system to regulate the expression of a heterologous gene, two separate plasmids must be introduced into the cells of interest. The introduction of the two plasmids into cells is performed using chemical or electrochemical procedures that are inherently inefficient in terms of the number of cells that take up the plasmid DNAs. The limitations of these DNA transfection procedures restrict the use of the two plasmid-based Tet system to cultured cells and can not be effectively used in vivo except in the case of transgenic animals (Furth, P. A., Onge, L. S., Boger, H., Gruss, P., Gossen, M., Kistner, A., Bujard, H & Hennighausen, L. (1994) Proc. Natl. Acad. Sci. USA. 91, 9302-9306; Fishman, G. I., Kaplan, M. L. & Buttrick, P. M. (1994) J. Clin. Invest. 93 1864-1868);
2) The original two plasmid-based Tet system (Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA, 89, 5547-5551) still shows gene expression when the system is switched OFF (W. P., unpublished data; Ackland-Berglund, C. E. & Leib, D. A. (1995) BioTechniques 18, 196-200). This means that the Tet system allows low level uninduced gene expression, which can be a significant problem in some applications